The spindle pole body of the pathogenic yeast Cryptococcus neoformans: variation in morphology and positional relationship with the nucleolus and the bud in interphase cells.

The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Cryptococcus neoformans was studied in detail by freeze-substitution and serial ultrathin sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar shaped. The dumbbell-shaped SPBs were 228-365 nm long with amorphous spheres on each end, each sphere being 78-157 nm in diameter. The bar-shaped SPBs were 103-260 nm long and 32-113 nm thick. They consisted of filamentous materials. The dumbbell-shaped SPBs were more frequent (61%) than the bar-shaped SPBs. The bar-shaped SPBs may be regarded as dumbbell-shaped SPBs whose spherical parts became sufficiently small. There seemed to be no relationship between the SPB shape and the cell cycle stage of G1-G2, since both types of SPB appeared not only in unbudded cells but also in budded cells and their appearance seems to be random. It is not clear at present whether morphological changes between dumbbell- and bar shapes have any physiological function. The SPB tended to be localized away from the nucleolus (141 degrees +/- 44 degrees), but localized randomly to the bud (97 degrees +/- 50 degrees). The present study highlights the necessity of observing a large number of micrographs in three dimensions to describe accurately the ultrastructure of the SPB in yeast.

Dynamics of the spindle pole body of the pathogenic yeast Cryptococcus neoformans examined by freeze-substitution electron microscopy.

Cryptococcus neoformans is an opportunistic human pathogen belonging to basidiomycetous fungi and has unique properties in cell cycle progression. In the present study, dynamics of the spindle pole body (SPB) during the cell cycle was examined using freeze-substitution and serial thin-sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar-shaped in G1 through G2 phases. At the beginning of prophase, globular elements of the SPB enlarged, associated with numerous cytoplasmic microtubules, and separated on the nuclear envelope. At prometaphase, the SPBs entered the nuclear region by breaking a part of the nuclear membrane, were located at the isthmus, and were associated with numerous nuclear microtubules. The nuclear division process was carried out in the daughter cell, though the nucleolus remained in the mother cell. At anaphase, one half of the nucleus returned to the mother cell. At telophase, the SPB element was extruded back to the cytoplasm from the nuclear region. By analyzing serial sections of 63 cells, duplication of the SPB was found to take place in the early G1 phase. Thus, the location, structure, and duplication cycle of the C. neoformans SPB are different from those of Saccharomyces cerevisiae, but have similarities to those of Schizosaccharomyces pombe.

Cytological study of cell cycle of the pathogenic yeast Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human pathogen belonging to basidiomycetous fungi and has unique properties in cell cycle progression. The purpose of this study was to measure the duration of the cell cycle in this yeast. Under standard liquid culture conditions (1% yeast extract, 1% polypeptone, and 1% glucose; 24 degrees C; and 150 rpm), the doubling time of exponentially growing C. neoformans was 132 +/- 16 min (mean +/- standard deviation), and the durations of the G1, S, G2, and M phases were about 71, 18, 25, and 18 min, respectively. DNA synthesis started before bud emergence, and finished by the time the size of the bud became 1/4 that of the mother cell. The doubling time of the daughter cells was about twice that of the mother cells. The spindle pole body was located on the outer nuclear envelope and showed a duplicated form from the G1 phase to the G2 phase. These data form a basis for further cell cycle study of C. neoformans.

Quantitative three-dimensional structural analysis of Exophiala dermatitidis yeast cells by freeze-substitution and serial ultrathin sectioning.

The morphologies, numbers, sizes and volumes of all organelles and cell components identified on ultrathin sections of aerobically grown exponential phase yeast cells of Exophiala dermatitidis in G1 phase were examined by freeze-substitution fixation and serial ultrathin sectioning. The cell wall consisted of three layers and occupied approximately 22% of the cell volume. The nucleus was approximately 1.8 microm in diameter and occupied approximately 7% of the cell volume. There was only one nucleolus in the nucleus and it occupied approximately 16% of the nuclear volume. There were 17-52 mitochondria per cell, occupying 7-12% of the cell volume. Five to ten endoplasmic reticula were present per cell; these occupied only 0.2% of the cell volume and did not form a network. There were 1-4 vacuoles per cell and they occupied 4-10% of the cell volume. Storage material was round and electron transparent and occupied 4-11% of the cell volume. The cytosol occupied 43-53% of the cell volume. The Golgi apparatus, spindle pole body, autophagosomes, multivesicular bodies, lipid bodies, microtubules and microfilaments occupied approximately 1% of the cell volume in total. About 200,000 ribosome particles, 1000 glycogen granules and several tens of microtubules (average length 0.78 microm) were present per yeast cell. The membranes of this yeast could be classified into three groups by their appearance and thickness. This is the first report, to our knowledge, that analysed all the components in the yeast cell quantitatively and in three dimensions, and provides fundamental information for understanding various aspects of cell biology.

Three-dimensional reconstruction of a pathogenic yeast Exophiala dermatitidis cell by freeze-substitution and serial sectioning electron microscopy.

The structure of a budding cell of the pathogenic yeast Exophiala dermatitidis was observed in three dimensions after freeze-substitution, serial ultrathin sectioning and computer reconstruction. The nucleus occupied about 10% of the cell volume. The spindle pole body was composed of two disk elements connected by an intervening midpiece, and occupied about 0.01% of the cell volume. The cell wall consisted of an inner transparent layer, a middle electron-opaque layer, and an outer fibrous layer. The mitochondria occupied about 10% of the cell volume. There were numerous mitochondria in the mother cell and the bud, but no 'giant mitochondrion' was seen. The ratio of mitochondrial volume within the bud to the mitochondrial volume of the cell was close to the ratio of bud:cell cytoplasmic volume. The results emphasize the importance of good cryofixation for 'perfect' preservation of yeast cell structure.

The spindle pole body duplicates in early G1 phase in the pathogenic yeast Exophiala dermatitidis: an ultrastructural study.

The spindle pole body of the pathogenic yeast Exophiala dermatitidis was observed during the cell cycle using freeze-substitution and serial ultrathin sectioning electron microscopy. The spindle pole body was located on the outer membrane of the nuclear envelope and consisted of two disk elements connected by an intervening midpiece in G1 through G2 phases. Each disk element was composed of filamentous materials and measured 150 nm in diameter and 100 nm in thickness. The midpiece had higher electron density and measured 60 nm in length and 40 nm in thickness. At the beginning of prophase, each disk element of the spindle pole body enlarged to more than double in size. They were separated on the nuclear envelope, and associated with numerous cytoplasmic microtubules. At mitosis, the spindle pole body entered the nuclear envelope, associated with numerous nuclear microtubules, and was located at the spindle poles. At the end of telophase, it was extruded back into the cytoplasm from the nuclear envelope. Three-dimensional analysis of cells in different cell cycles suggested that duplication of the spindle pole body took place in early G1 phase. Thus, the location, structure, and duplication cycle of the E. dermatitidis spindle pole body were different from those of Saccharomyces cerevisiae.

Electron microscopy of pathogenic yeasts Cryptococcus neoformans and Exophiala dermatitidis by high-pressure freezing.

A high-pressure freezing method was used to observe the ultrastructure of pathogenic yeasts, Cryptococcus neoformans and Exophiala dermatitidis, after freeze-substitution and ultrathin sectioning. The method well preserved the cell structure in its natural state, since the capsule, cell wall, plasma membrane, nucleus, outer and inner nuclear membranes, nuclear pores, nucleolus, mitochondria, mitochondrial membrane and cristae, vacuoles, endoplasmic reticulum, Golgi apparatus, spindle pole body, ribosomes, lipid droplets, microtubules, actin filaments, and glycogen granules were clearly visible. The method was shown to freeze cells as deep as 0.1 mm by sectioning the sample perpendicular to specimen surface. The quality of the cell image was similar to that obtained by a rapid freezing method when compared using the same materials. Thus, high-pressure freezing would be useful for making serial ultrathin sections for three-dimensional analysis of cells, which should give basic information of structure and function of pathogenic yeast cells necessary for finding an effective therapy for mycoses.